The EF-hand family of Ca2+ binding proteins has a central role in many aspects of Ca2+ signal transduction pathways, from the regulation of the opening and closing of channels, to controlling the intensity and duration of Ca2+ signals, to the transduction of these signals into biochemical and biomechanical responses. Over 1000 EF-hand protein motifs have been identified from their unique sequence signatures. Yet despite their widespread distribution, very little is known about what features of their sequences provide them with their specific functions or what molecular mechanisms are used to distinguish among their diverse cellular targets. The objective of our research program is to define the molecular basis for the biochemical and biological function of EF-hand proteins, so that we may better understand their roles in health and disease. Ultimately, we will use this knowledge to alter or de novo design Ca2+-dependent cellular activities for medical and biotechnology applications. The overall objective of the current proposal is to answer two key questions regarding the underlying molecular basis for intracellular Ca2+ signal transduction: (i) How does the sequence of an EF-hand protein specify its response to the binding of Ca2+? (ii) How do different EF-hand proteins interact with and modulate specific protein targets? The first question will be addressed by a series of coupled mutagenesis, biophysical, and structural studies using calbindin D9k as a model system. A key goal in this work will be to create a new calbindin-calmodulin hybrid protein, calbindomodulin. The second question will be addressed by combined biophysical and structural studies seeking to determine the mechanisms of the Ca2+-induced activities of caltractin and the regulatory domain of the calcium dependent protein kinase. These results will link the large volume of experimental data available on EF-hand proteins, uncover the mechanisms by which these proteins function, and help to define their biological activities.